Selective Bcl-2 Inhibitor Venetoclax Research Articles

Abstract 5991: Improved venetoclax therapy by a novel conjugate with artemisinin by NOXA-mediated reduction of Mcl-1 and cyclin D1 protein in myeloid leukemia cells

Abstract Evasion of apoptosis is crucial for the growth, survival and chemoresistance of myeloid leukemia. The Bcl-2 selective inhibitor venetoclax is the only apoptosis inducer approved for clinical use. Venetoclax in combination with hypomethylating agents or low-dose cytarabine has now become the standard care for elderly AML patients. However, a sizeable fraction of patients are either refractory to venetoclax combination therapy or ultimately relapse. High or induced expression of Mcl-1 and Bcl-xL mediates venetoclax resistance. Previously we reported that artemisinin enhances venetoclax apoptosis induction by degrading Mcl-1 through NOXA induction. We designed a novel conjugate A1 of venetoclax with dihydroartemisinin. A1 shows dual functions of inhibiting Bcl-2 and inducing NOXA. A1 treatment releases Bim from both Bcl-2 and Mcl-1, further degrades Mcl-1 protein, and presents ten-fold stronger apoptotic induction ability in venetoclax insensitive cells. Acquired increase of Mcl-1 protein was observed in venetoclax resistant MOLM-13/VEN cells, which are sensitive to A1-induced apoptosis. In Bcl-xL expressing venetoclax refractory leukemia cells A1 obtains a new mechanism of inhibiting cell cycling by downregulating cyclin D1 through a new NOXA/cyclin D1 cascade, mediated through the endoperoxide moiety of artemisinin and intracellular iron, independent of Bcl-2 inhibition. We reveal a new compound A1 overcoming venetoclax resistance by NOXA-mediated degradation of Mcl-1 and cyclin D1 protein. Citation Format: Jingyi Zhang, Zhenwei Zhang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. Improved venetoclax therapy by a novel conjugate with artemisinin by NOXA-mediated reduction of Mcl-1 and cyclin D1 protein in myeloid leukemia cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5991.

Abstract P29: Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth

Abstract New targeted therapies such as the selective and potent BCL-2 inhibitor Venetoclax have revolutionized the treatment of acute myeloid leukemia (AML). However, due to the cellular heterogeneity of the disease, drug-resistant cell types often emerge and drive relapse. We found that the eukaryotic translation initiation factor 4A (EIF4A) is highly expressed in multiple primitive AML cell types compared to their healthy counterparts, suggesting its potential as a therapeutic target. By unwinding complex structures in the 5’ UTR of transcripts, eIF4A promotes the translation of oncogene transcripts. A first-in-class drug, Zotatifin, inhibits the helicase activity of eIF4A and curtails tumor growth in receptor tyrosine kinase-driven tumors. Zotatifin is currently being evaluated in phase 1/2 trials across multiple solid tumors (NCT04092673). However, its effectiveness in blood cancers such as AML remains unexplored, and it is unknown whether Zotatifin may work synergistically with Venetoclax. Here, we treated AML cell lines in vitro and primary healthy or AML bone marrow ex vivo with Zotatifin or Venetoclax alone and in combination, and demonstrate synergistic killing of AML cells by the combination therapy (ZIP synergy score = 57.0 in MOLM-13, 19.3 in MV4-11, P < 0.0001). Mechanistically, we show that Zotatifin decreases eIF4A and AKT expression, and its combination with Venetoclax drives increased BAK and cleaved BAK expression, as well as increased cleavage of MCL-1 and BCL-2, consistent with drug synergy by convergence on the apoptotic pathway. In pilot colony-forming unit (CFU) assays, we found the combination to be tolerable in healthy CD34+ human hematopoietic stem and progenitor cells (HSPCs). To test drug efficacy in vivo, we generated a patient-derived xenograft (PDX) AML model from a patient who experienced relapse with complex cytogenetics, FLT3 and NPM1 mutations. Only the combination of Zotatifin and Venetoclax but not monotherapies effectively suppressed tumor burden as assessed by human CD45 engraftment in the peripheral blood. Further, the combination of Zotatifin and Venetoclax significantly prolonged survival of xenografted mice (median survival after treatment: vehicle 11 days, Venetoclax 13 days, Zotatifin 15 days, combination 42 days, P = 0.0002). Together, our data indicates that Zotatifin is synergistic with Venetoclax and may represent an effective treatment option for patients who are either primary refractory or have developed resistance to existing Venetoclax-containing regimens. Citation Format: Yoke Seng Lee, Jonathan Good, Noha Awais, Benjamin Eschle, Jenny Noel, Jean Acosta, Erica DePasquale, David Cucchi, Prafulla Gokhale, Fadi Najm, Peter van Galen. Zotatifin, the eukaryotic translation initiation factor 4A (eIF4A) inhibitor, synergizes with Venetoclax to inhibit acute myeloid leukemia cell growth [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P29.

Selective pharmacologic targeting of CTPS1 shows single-agent activity and synergizes with BCL2 inhibition in aggressive mantle cell lymphoma.

Innovative therapeutic strategies have emerged over the past decade to improve outcomes for most lymphoma patients. Nevertheless, the aggressive presentation seen in high-risk mantle cell lymphoma (MCL) patients remains an unmet medical need. The highly proliferative cells that characterize these tumors depend on nucleotide synthesis to ensure high DNA replication and RNA synthesis. To take advantage of this vulnerability, STP-B, a clinically available small molecule selectively targeting CTP synthase 1 (CTPS1) has been recently developed. CTPS1 is a key enzyme of the pyrimidine synthesis pathway mediated through its unique ability to provide enough CTP in highly proliferating cells. Herein, we demonstrated that CTPS1 was expressed in all MCL cells, and that its high expression was associated with unfavorable outcomes for patients treated with chemotherapy. Using aggressive MCL models characterized by blastoid morphology, TP53 mutation or polyresistance to targeted therapies, we showed that STP-B was highly effective at nanomolar concentrations in vitro and in vivo, irrespective of these high-risk features. Inhibition of CTPS1 rapidly leads to cell cycle arrest in early S-phase accompanied by inhibition of translation, including of the anti-apoptotic protein MCL1. Consequently, CTPS1 inhibition induced synergistic cell death in combination with the selective BCL2 inhibitor venetoclax, both in vitro and in vivo. Overall, our study identified CTPS1 as a promising target for MCL patients and provided a mechanism-based combination with the BCL2 inhibitor venetoclax for the design of future chemotherapy-free treatment regimens to overcome resistance.

MALT1 Protease Inhibition Overcomes BTK Inhibitor Resistance and Shows Synergistic Activity with Venetoclax in Models of B Cell Lymphoma and Leukemia

Background Specific B cell malignancies, including the aggressive non-germinal center B cell-like (GCB) subtype of diffuse large B cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL), are driven by constitutive activation of the transcription factor NF-kB. Chronic signaling resulting from mutations or antigen-induced activation of the B cell receptor (BCR) pathway drives NF-kB activity in these tumors resulting in sustained proliferation and survival pathways. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) represents a key mediator of the BCR/NF-kB signal transduction pathway responsible for maintaining survival and driving proliferation of NF-kB-addicted B cell tumors by proteolytically cleaving NF-kB antagonists. Aims Pharmacological inhibition of MALT1 protease activity may provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Methods A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results ABBV-MALT1 potently inhibits MALT1 activity in vitro while showing high selectivity against a panel of proteases, kinases and other types of receptors and enzymes. Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-kB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton's tyrosine kinase (BTK) inhibitors. NF-kB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-kB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Summary/Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with DLBCL, CLL and other B cell malignancies. Disclosures All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Effective venetoclax-based treatment in relapsed/refractory multiple myeloma patients with translocation t(6;14).

Introduction: The selective Bcl-2 inhibitor venetoclax has shown promising therapeutic potential in multiple myeloma, particularly in cases associated with t(11;14) IGH::CCND1 translocation. However, the efficacy of venetoclax in myeloma patients with the t(6;14) IGH::CCND3 translocation remains less investigated. Methods: In this study, we conducted a retrospective analysis to investigate the efficacy of venetoclax-based therapy in relapsed/refractory myeloma patients with t(6;14) translocation. The treatment courses of three patients, that included previous therapies and responses to venetoclax, were assessed. Clinical data, laboratory results, and adverse events were analyzed to evaluate treatment outcomes. Results: Our findings demonstrated remarkable therapeutic responses in three consecutive patients with t(6;14) translocation-associated myeloma who received venetoclax-based therapy. Patient 1, a lenalidomide-bortezomib-daratumumab and alkylator treatment refractory patient, achieved sustained stringent complete remission (sCR) after combining carfilzomib-dexamethasone with venetoclax, which was his best response ever. Similarly, Patient 2, refractory to frontline bortezomib-thalidomide-dexamethasone therapy, attained CR following a transition to bortezomib-dexamethason-venetoclax treatment. Patient 3, who was immunomodulatory (IMID)-intolerant, showed a highly favorable response to venetoclax-dexamethasone therapy after his first relapse following autologous stem cell transplantation. No significant adverse effects were observed in any of the patients. Discussion: Our study provides compelling preliminary evidence for the efficacy of venetoclax in t(6;14) translocation-associated myeloma. The outcomes observed in our patients suggest that venetoclax-based therapy holds substantial promise as an effective treatment option for this specific genetic subgroup. Furthermore, the similarities in treatment response between t(11;14) and t(6;14) translocation subgroups highlight the importance of personalized approaches targeting specific genetic abnormalities to optimize therapeutic outcomes.

The comprehensive expression of BCL2 family genes determines the prognosis of diffuse large B-cell lymphoma

Diffuse large B-cell lymphoma (DLBCL) is a prevalent and aggressive non-Hodgkin's lymphoma, and 40% of patients succumb to death. Despite numerous clinical trials aimed at developing treatment strategies beyond the conventional R–CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) regimen, there have been no positive results thus far. Although the selective BCL2 inhibitor venetoclax has shown remarkable efficacy in chronic lymphocytic leukemia, its therapeutic effect in DLBCL was limited. We hypothesized that the limited therapeutic effect of venetoclax in DLBCL may be attributed to the complex expression and interactions of BCL2 family members, including BCL2. Therefore, we aimed to comprehensively analyze the expression patterns of BCL2 family members in DLBCL. We analyzed 157 patients with de novo DLBCL diagnosed at Asan Medical Center and Ajou University Hospital. The mRNA expression levels of BCL2 family members were quantified using the NanoString technology. BCL2 family members showed distinct heterogeneous expression patterns both intra- and inter-patient. Using unsupervised hierarchical cluster analysis, we were able to classify patients with similar BCL2 family expression pattern and select groups with clear prognostic features, C1 and C6. In the group with the best prognosis, C1, the expression of pro-apoptotic and pro-apoptotic BH3-only group gene expressions were increased, while anti-apoptotic group expression was significantly increased in both C1 and C6. Based on this, we generated the BCL2 signature score using the expression of pro-apoptotic genes BOK and BCL2L15, and anti-apoptotic gene BCL2. The BCL2 signature score 0 had the best prognosis, score 1/2 had intermediate prognosis, and score 3 had the worst prognosis (EFS, p = 0.0054; OS, p = 0.0011). Multivariate analysis, including COO and IPI, showed that increase in the BCL2 signature score was significantly associated with poor prognosis for EFS, independent of COO and IPI. The BCL2 signature score we proposed in this study provides information on BCL2 family deregulation based on the equilibrium of pro-versus anti-apoptotic BCL2 family, which can aid in the development of new treatment strategies for DLBCL in the future.

Abstract 6156: MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia

Abstract Introduction: Specific B cell malignancies, including CLL and the aggressive non-GCB subtype of DLBCL, are driven by constitutive activation of the B cell receptor (BCR) pathway and the transcription factor NF-κB. Pharmacological inhibition of MALT1 protease, a key mediator of the BCR/NF-κB signal transduction pathway, may therefore provide an attractive treatment option for patients with these cancers. Further, as combination therapy is often required for the treatment of aggressive B cell malignancies, the identification of therapies that synergistically combine with MALT1 inhibitors could afford additional and promising treatment options. Experimental Procedures: A highly potent and orally bioavailable MALT1 protease inhibitor (ABBV-MALT1) was used to test the hypothesis that MALT1 inhibition will abrogate the proliferation of preclinical models of B cell malignancies in vitro and in vivo. Tumors treated with ABBV-MALT1 were subjected to transcriptomic and functional proteomic assays to elucidate molecular mechanisms of action and rational combination partners. Results: Mechanistic studies reveal that ABBV-MALT1 effectively inhibits signal transduction of the BCR pathway and reduces NF-κB gene activation in non-GCB DLBCL cell lines resulting in cell cycle arrest and diminished viability. In vivo, oral administration of this compound demonstrates robust tumor growth inhibition in several models of B cell tumors, including non-GCB DLBCL models that are resistant to Bruton’s tyrosine kinase (BTK) inhibitors. NF-κB target genes include the pro-survival family members BCL-XL and BCL2-A1, which aid in regulation of the intrinsic apoptosis pathway. As ABBV-MALT1-induced inhibition of the NF-κB pathway resulted in downregulation of these genes, we hypothesized that the associated tumor models would become increasingly dependent on the pro-survival family member BCL-2. To test this hypothesis, combination studies of ABBV-MALT1 and the selective BCL-2 inhibitor venetoclax were performed in both cell line and patient-derived xenograft models of DLBCL. Herein we show that concomitant administration of ABBV-MALT1 and venetoclax results in dramatic antitumor activity in all models tested in vivo. This efficacy also translates to primary patient CLL cells in vitro where the combination confers greater levels of apoptosis compared to either agent alone. Conclusion: ABBV-MALT1 demonstrates robust single agent anti-tumor activity in malignant B cell models that are resistant to BTK inhibitors. Moreover, combination of ABBV-MALT1 with the BCL-2 inhibitor venetoclax shows synergistic cell killing of B cell tumors in vitro and dramatic tumor regression in vivo. Together, these data indicate that MALT1 inhibition may overcome BTK inhibitor resistance and combine with venetoclax to effectively treat patients with B cell malignancies. Citation Format: Joshua P. Plotnik, Raghuveer S. Mali, Velitchka Bontcheva, Colleen Dowell, Jun Chen, Adam E. Richardson, Ryan A. McClure, Paul Jung, Lan Pham, Andrew J. Souers, Jonathan A. Meulbroek, William N. Pappano. MALT1 protease inhibition overcomes BTK inhibitor resistance and shows synergistic activity with venetoclax in models of B cell lymphoma and leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6156.

Abstract 6150: AZD0466, a dual BCL-2/XL targeting nanomedicine, is active in small cell lung cancer models

Abstract Small cell lung cancer (SCLC) is an aggressive malignancy with critical need for new therapies. While currently treated as a single disease, SCLC is heterogenous, comprised of several transcriptional subtypes. Each of these subtypes has distinct drivers and may warrant unique therapeutic targets. Two potential therapeutic targets for SCLC are the pro-survival proteins BCL-2 and BCL-XL. BCL-2 is overexpressed in ASCL1 (A) and POUF3 (P) subtypes of SCLC. We therefore sought to evaluate efficacy of the dual BCL-2/XL inhibitor AZD0466 in SCLC models and to determine whether transcriptional subtype would predict response. AZD0466 is a novel drug-dendrimer conjugate. The active moiety, AZD4320, is a potent dual inhibitor of BCL-2 and BCL-XL. AZD4320 is covalently conjugated to a 5th-generation PEGylated poly-lysine dendrimer through a hydrolytically labile linker to make AZD0466. AZD0466 has been optimized to deliver efficacy while mitigating potential Cmax-driven on-target toxicities of AZD4320. AZD4320 was active (IC50 ≤0.1 µM) in 9/27 SCLC cell lines. AZD4320 in vitro sensitivity was enriched in cell lines that represented A and P subtypes of SCLC compared to NEUROD1 and YAP1 subtypes. We next profiled AZD0466 in a panel of SCLC patient-derived models: 14 patient-derived xenografts and 10 circulating tumor cell-derived xenografts. AZD0466 monotherapy dosed weekly IV was active in 12/24 SCLC xenografts, driving regressions in 8 models. AZD0466 drove efficacy and cleaved caspase-3 induction in a dose-dependent manner. Similar to in vitro, AZD0466 in vivo efficacy was enriched in subtype-A, driving responses in 10/14 ASCL1 models (7 regression, 3 stable disease). AZD0466 response also correlated strongly with BCL-2 mRNA expression (P<0.0001). AZD0466 outperformed the selective BCL-2 inhibitor venetoclax in 6/10 models. Notably, AZD0466 was active in models resistant to platinum/etoposide chemotherapy, the standard-of-care for SCLC. Together, these data suggest BCL-2/XL inhibition has therapeutic potential in SCLC. AZD0466 is in clinical development. The first-in-human study treated 9 patients with advanced solid tumors (NCT04214093) at doses from 50-200mg, all of which were well-tolerated. The BOR was SD observed in 3 patients (100mg) with 1 patient receiving treatment for 5.5 months. AZD0466 is now under evaluation in patients with hematologic malignancies (NCT04865419 and NCT05205161). AZD0466 has been dosed in 33 patients up to 2400mg. No DLTs have been reported to date. Initial clinical activity has been observed through reduction of bone marrow blasts following AZD0466 treatment. AZD0466 exhibits linear PK, consistent across solid tumor and leukemia patients. The doses tested are in line with preclinical studies in SCLC. Citation Format: Courtney L. Andersen, Giulia Fabbri, David Jenkins, Zumla Cader, Shringi Sharma, Areya Tabatabai, Srividya Balachander, Jordan Roebuck, Melanie Galvin, Kathryn Simpson, Caroline Dive, Jordi Rodon Ahnert, Jamal Saeh. AZD0466, a dual BCL-2/XL targeting nanomedicine, is active in small cell lung cancer models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6150.

Landscape of BCL2 Resistance Mutations in a Real-World Cohort of Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia Treated with Venetoclax

The oral, highly selective Bcl2 inhibitor venetoclax has substantially improved the therapeutic landscape of chronic lymphocytic leukemia (CLL). Despite the remarkable response rates in patients with relapsed/refractory (R/R) disease, acquired resistance is the leading cause of treatment failure, with somatic BCL2 mutations being the predominant genetic drivers underpinning venetoclax resistance. To assess the correlation between disease progression and the most common BCL2 mutations G101V and D103Y, sensitive (10-4) screening for the most common BCL2 mutations G101V and D103Y was performed in 67 R/R CLL patients during venetoclax single-agent or venetoclax-rituximab combination therapy. With a median follow-up time of 23 months, BCL2 G101V and D103Y were detected in 10.4% (7/67) and 11.9% (8/67) of the cases, respectively, with four patients harboring both resistance mutations. Ten out of eleven patients carrying BCL2 G101V and/or D103Y experienced relapse during the follow-up period, representing 43.5% of the cases (10/23) showing clinical signs of disease progression. All BCL2 G101V or D103Y variants were detected in patients receiving venetoclax as a continuous single-agent treatment while these mutations were not observed during or after fixed-duration venetoclax therapy. Targeted ultra-deep sequencing of BCL2 uncovered three additional variants in four patient samples obtained at relapse, suggesting convergent evolution and implying a cooperating role of BCL2 mutations in driving venetoclax resistance. This cohort is the largest R/R CLL patient population reported to date in which BCL2 resistance mutations were investigated. Our study demonstrates the feasibility and clinical value of sensitive screening for BCL2 resistance mutations in R/R CLL.

A novel Mcl-1 inhibitor synergizes with venetoclax to induce apoptosis in cancer cells

BackgroundEvading apoptosis by overexpression of anti-apoptotic Bcl-2 family proteins is a hallmark of cancer cells and the Bcl-2 selective inhibitor venetoclax is widely used in the treatment of hematologic malignancies. Mcl-1, another anti-apoptotic Bcl-2 family member, is recognized as the primary cause of resistance to venetoclax treatment. However, there is currently no Mcl-1 inhibitor approved for clinical use.MethodsPaired parental and Mcl-1 knockout H1299 cells were used to screen and identify a small molecule named MI-238. Immunoprecipitation (IP) and flow cytometry assay were performed to analyze the activation of pro-apoptotic protein Bak. Annexin V staining and western blot analysis of cleaved caspase 3 were employed to measure the cell apoptosis. Mouse xenograft AML model using luciferase-expressing Molm13 cells was employed to evaluate in vivo therapeutic efficacy. Bone marrow samples from newly diagnosed AML patients were collected to evaluate the therapeutic potency.ResultsHere, we show that MI-238, a novel and specific Mcl-1 inhibitor, can disrupt the association of Mcl-1 with BH3-only pro-apoptotic proteins, selectively leading to apoptosis in Mcl-1 proficient cells. Moreover, MI-238 treatment also potently induces apoptosis in acute myeloid leukemia (AML) cells. Notably, the combined treatment of MI-238 with venetoclax exhibited strong synergistic anti-cancer effects in AML cells in vitro, MOLM-13 xenografts mouse model and AML patient samples.ConclusionsThis study identified a novel and selective Mcl-1 inhibitor MI-238 and demonstrated that the development of MI-238 provides a novel strategy to improve the outcome of venetoclax therapy in AML.

Hexamethylene Amiloride Sensitizes AML to Venetoclax through Inducing Lysosomal Membrane Permeabilization

Chemotherapy remains the standard therapeutic approach for acute myeloid leukemia (AML). Although several new drugs have received approval for clinical usage, including the selective BCL2 inhibitor venetoclax, refractory disease or resistance continues to be a recurring theme, and exploring more effective therapies for AML treatment remains necessary. Intracellular pH dynamics are vital determinants for cancer progression and chemoresistance. Tumors maintain a more alkaline intracellular pH (pHi) dependent on proton exporters, including sodium-hydrogen exchangers (NHEs). Elevated NHE1 expression has emerged as a biomarker for the tumorigenesis and prognosis of solid tumors. However, whether targeting NHE1 with inhibitors could inhibit AML cell growth and the underlying mechanisms remain unknown. We found that NHE1 was highly expressed in AML and predicted a worse prognosis based on data analysis of public database. Moreover, NHE1 knockdown inhibited the growth of AML cells. In addition, NHE1 inhibitor Hexamethylene amiloride (HA) significantly inhibited proliferation and promoted apoptosis in a concentration-dependent manner in both AML cell lines and primary AML cells. Next, we combined HA with several first-line or novel drugs and found that HA combined with venetoclax significantly inhibited viability of AML cells. AML patients with higher NHE1 expression were less sensitive to BCL-2 inhibitors. This suggested that HA and venetoclax might be an effective combination to treat AML. As expected, HA synergized with venetoclax to inhibit proliferation and promote apoptosis. In vivo potential of HA combined with venetoclax was evaluated in a MV4-11-derived xenograft mouse model (Fig.1). The combinational regimen prolonged survival and alleviated leukemia burden in bone marrow, spleen and peripheral blood. We further validated the effect of HA with venetoclax with primary AML cells. Consistent with results in cell lines, HA combined with venetoclax also exerted synergistic effect in inhibiting viability and promoting apoptosis. RNA-Seq analysis revealed that lysosome pathway was significantly enriched in the combination group. We further verified the increased quantity of lysosome, and the swelling and rupture of lysosome with TEM (Fig.2). Moreover, HA combined with venetoclax induced obvious LGALS3 puncta formation in this cell line, which represented the occurrence of lysosomal membrane permeabilization (LMP), leading to the release of proteases which eventually results in cell death. In conclusion, our results for the first time provide evidence that the NHE1 inhibitor HA or its combination with venetoclax efficiently inhibits the growth of AML in vitro and in vivo. This study might shed light on the clinical treatment of AML involving proton exporter inhibitors with venetoclax as combinatory regime. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Biomarkers of Clinical Resistance to Decitabine and Venetoclax Therapy Inferred By Transcriptome and Methylation Analyses

The combination of the selective BCL-2 inhibitor venetoclax (VEN) with the hypomethylating agent (HMA) decitabine (DEC) is highly effective in treating older ineligible for intensive chemotherapy AML patients. However, approximately 15-34% of newly diagnosed (ND) and 60% of relapsed/refractory (R/R) patients fail to achieve complete remission (CR) (DiNardo et al. New Engl J Med. 2020, DiNardo et al. Lancet Haematol. 2020). Factors that reduce efficacy remain to be elucidated. To dissect underlying mechanisms of HMA-VEN resistance, we performed RNA-seq utilizing primary samples from patients treated on a prospective clinical trial of 10-day DEC with VEN (NCT03404193). Previously, we reported that alterations of transcriptome signatures of energy-metabolism and cell migration/adhesion pathways were upregulated in primary resistant ND group, and immune-system-associated genes were downregulated in R/R group (Yamatani et al. ASH 2021). This study investigated transcriptomic and DNA methylation signatures associated with DEC10-VEN response or resistance in ND and R/R AML. Because DEC was reported to strongly induce interferon-stimulated genes (ISGs) through gene methylation modifications, we focused on 116 ISGs. An integrated analysis of DNA methylation status around the transcription start site (TSS) was performed using the Infinium methylated EPIC array (Illumina). Forty-four pre-treatment samples from 32 patients with AML from DEC-VEN clinical trial, ND patient group (CR/CRi 14; non-responder, NR 11) and R/R patient group (CR/CRi 6; NR 13), were analyzed. First, we performed the comparative analysis of NR vs CRs. In the ND group, NR cases showed significantly higher gene expression and lower TSS methylation of an urea cycle enzyme, ASS1 (Argininosuccinate Synthase 1) with RNA-seq Log2 fold-change (FC) 3.6 and methylation array Δβ (differential methylation β-value) -0.11, p < 0.05, and of an actin-based molecular motor, MYO1C (Myosin 1C), Log2FC 2.5, Δβ = -0.18, p< 0.05, than in CR cases. In the R/R group, gene expression levels of seven ISGs, including CCL3 (C-C motif chemokine 3), were significantly lower in NR than in CRs, with no difference in methylation status. Next, changes in gene expression levels and TSS methylation status after DEC-VEN treatment were investigated in non-responders in ND group (pre 19, post 12) and R/R group (pre 16, post 4). After DEC-VEN treatment, PPARG (Peroxisome Proliferator-Activated Receptor γ), a transcription factor that activates fatty acid metabolism and negatively regulates subsets of IFN-γ target genes, was significantly upregulated along with its TSS demethylation both in ND (Log2FC 4.9, Δβ = -0.14, p < 0.05), and R/R groups (Log2FC 3.7, Δβ = -0.06, p < 0.05). CDC42EP4, an effector protein of CDC42 involved in the organization of the actin cytoskeleton, was upregulated after the treatment in the R/R group (Log2FC 2.7, Δβ = -0.14, p < 0.05). Based on these findings, we validated transcriptional and methylation changes of ASS1, PPARG, MYO1C and CDC42EP4 by RT-qPCR and bisulfite pyrosequencing using HL60 and MV4-11 AML cell lines in which DEC (IC50, HL60, 249 nM; MV4-11, 2090 nM) and VEN (IC50, HL60, 18 nM; MV4-11, 64 nM) produced additive or synergistic combination effect. Cells were treated by DEC and/or VEN under BM-derived mesenchymal stem cells (MSCs) co-culture conditions to mimic the BM microenvironment. DEC upregulated gene expression levels with TSS demethylation of PPARG, MYO1C and CDC42EP4 both in HL60 and MV4-11, and of ASS1 only in HL-60. VEN treatment alone or in combination with DEC did not induce significant changes in the expression of these tested genes. Of note, PPARG expression was further upregulated under MSCs co-culture condition without TSS methylation status change in AML cells. DEC treatment induced no significant changes of PPARG in MSC. These findings suggest that not only TSS demethylation but also interaction with BM stromal cells may be involved in PPARG upregulation associated with energy production in AML cells. Taken together, DEC-VEN treatment stimulates two key factors, energy metabolism and interaction with the BM microenvironment, which may reduce therapeutic efficacy by increasing gene expression of PPARG,MYO1C,ASS1, and CDC42EP4 through TSS demethylation and/or interaction with BM stromal cells. Experimentally, the consequences of overexpression / knockdown of these genes will be further validated.

Triple targeted therapy is a new perspective treatment strategy for patients with FLT3-mutated acute myeloid leukemia. the literature review and single-centre experience

Internal tandem duplication is the most common form of mutation in FMS-like tyrosine kinase 3 (FLT3) in different haematological malignancies, highlighting in acute myeloid leukaemia (AML) and is associated with increased risk of relapse and reduced overall survival. A major breakthrough in the treatment of FLT3-mutated AML has been achieved through the use of highly selective FLT3 tyrosine kinase inhibitors, both in monotherapy and in combination with standard intensive cytotoxic chemotherapy. The desire to improve the outcomes of patients with AML, including those with relapse and refractory disease, has led to attempts to use non-standard therapeutic options. Enhancement of the antileukemic effects of the second-generation FLT3 inhibitor Gilteritinib may be achieved through synergy with the hypomethylating agent 5-azacytidine and the selective Bcl-2 inhibitor Venetoclax. Thus, targeted triple therapy is a promising option in the treatment of patients with FLT3-mutated AML. This study sought to evaluate the effectiveness of “triple therapy” regimen in 4 patients with relapsed/refractory FLT3 mutated AML. We found that the use of this combination showed rapid response with good safety and frequently allowed subsequent transplant and achieve durable clinical benefit.

P561: VIALE-T: A RANDOMIZED, OPEN-LABEL, PHASE 3 STUDY OF VENETOCLAX IN COMBINATION WITH AZACITIDINE AFTER ALLOGENEIC STEM CELL TRANSPLANTATION IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

Background: Acute myeloid leukemia (AML) is an aggressive malignancy and is the most common and second most common form of acute leukemia in adults and children, respectively. The combination of the highly selective BCL-2 inhibitor venetoclax and the hypomethylating agent azacitidine was shown to be safe and effective in clinical trials (DiNardo et al. Blood. 2019;133:7-17; DiNardo et al. N Engl J Med. 2020;383:617-629) and is approved by the United States Food and Drug Administration and European Medicines Agency for the treatment of patients with AML who are not eligible to receive intensive chemotherapy. Following allogeneic stem cell transplantation (alloSCT), most patients do not receive antileukemic therapy; however, an unmet need remains as disease relapse and graft-versus-host disease (GvHD) commonly occur posttransplant. In addition to the antileukemic effect of venetoclax shown in clinical studies, preclinical studies suggest venetoclax may mitigate the risk of GvHD. VIALE-T is a Phase 3, randomized, open-label trial in progress (NCT04161885) evaluating the safety and efficacy of venetoclax in combination with azacitidine versus best supportive care (BSC) as maintenance therapy following alloSCT in patients with AML. Aims: N/A Methods: This Phase 3 study consists of 2 parts (Figure). Key inclusion criteria include diagnosis of AML; plans to receive alloSCT or have received alloSCT within the past 30 days; bone marrow blasts <10% before pretransplant conditioning and <5% posttransplant; have received myeloablative, or reduced intensity, or nonmyeloablative pretransplant conditioning protocols. Grafts are allowed from various sources (bone marrow, peripheral blood stem cells, cord blood cells). Patients must be ≥18 years old for Part 1 and ≥12 years old for Part 2. Additionally, patients must meet key laboratory values for absolute neutrophil count (Part 1, ≥1500/µL; Part 2, ≥1000/µL), platelet count (Part 1, ≥80,000/µL; Part 2, ≥50,000/µL), bilirubin ≤3 times the upper limit of normal, and creatinine clearance >30 mL/min. Patients who have received venetoclax and had no history of disease progression while receiving venetoclax are eligible. Part 1 evaluates dose levels of venetoclax combined with azacitidine to determine the recommended Phase 3 dose (RP3D), which will be confirmed in approximately 12 additional patients enrolled in the Safety Expansion Cohort. Part 2 will be a randomized, open-label evaluation of the RP3D of venetoclax combined with azacitidine and BSC versus BSC only in adults and children aged 12 years or older. All venetoclax-treated patients will receive antibiotic prophylaxis during Cycle 1. The primary endpoint for Part 1 is the frequency of dose-limiting toxicities. The primary endpoint for Part 2 is relapse-free survival as assessed by an independent review committee. Key secondary endpoints for Part 2 include overall survival, GvHD-free relapse-free survival, and the rate of patients without higher grade GvHD at 90 days after randomization. Enrollment into the Safety Expansion Cohort will be completed in 2021. Part 2 will enroll approximately 400 patients across approximately 175 participating study sites in 17 countries, with recruitment beginning in 2022. Results: N/A Image:Summary/Conclusion: N/A

P344: THE DUAL BCL-2 AND BCL-XL INHIBITOR AZD4320 SHOWS ON-TARGET ACTIVITY IN ALL AND ACTS SYNERGISTICALLY WITH MCL-1 INHIBITION

Background: Targeting anti-apoptotic BCL-2 family proteins by BH3-mimetics has become a promising treatment strategy in acute lymphoblastic leukemia (ALL). Heterogeneous activity of the selective BCL-2 inhibitor venetoclax has been observed, but other anti-apoptotic proteins including BCL-XL promote venetoclax insensitivity. In clinical trials, targeting BCL-XL has previously resulted in a dose-limiting decrease of platelets. AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia. Aims: In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845). The on-target activity of the inhibitors was functionally characterized and combination effects were analyzed. Methods: Cell viability assays were performed in ALL cell lines and patient-derived xenograft (PDX) samples analyzing half maximal effective concentrations (EC50). Protein complexes and expression of apoptosis regulators were analyzed by immunoprecipitation and immunoblotting. Dynamic BH3 profiling using synthetic BH3-peptides was performed to determine the dependency of ALL cells on BCL-2 family proteins. Combination effects were assessed by dose-response matrix analyses. Results: First, we determined the efficacy of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of the MCL-1 inhibitor AZD5991 for cell death induction in seven B-cell precursor (BCP) ALL cell lines and in a series of 13 PDX samples, showing heterogeneous responses. Interestingly, sensitivities of individual samples to both compounds were not associated with each other. However, we found a significant correlation of sensitivity to the dual inhibitor (AZD4320) with BCL-2 inhibition (venetoclax; N=13; rs=0.56; p=0.049), but no association with BCL-XL inhibition (A-1331852). Analyzing activities of all five BH3-mimetics including venetoclax, we found lowest EC50 values for AZD4320, indicating particular sensitivity for this dual inhibitor as compared to the single inhibitors (p<0.001). Investigating dependencies of ALL cells on BCL-2 family proteins by dynamic BH3 profiling, we found a shift towards MCL-1-dependence upon exposure to AZD4320, while AZD5991 induced an increased combined dependence on BCL-2 and BCL-XL, indicating potential synergistic activity of triple inhibition. Using co-immunoprecipitation analyses, we found that the exposure of ALL cells to AZD4320 reduced binding of both BCL-2 and BCL-XL to BIM, confirming on-target activity. Moreover, AZD5991 reduced binding of BIM to MCL-1. Accordingly, combined treatment with both inhibitors results in the release of BIM and downstream apoptosis signaling. Assessing combinatorial treatment in a primary PDX sample using multi-dose matrix analyses of both inhibitors revealed a positive mean Bliss synergy score of +4.8 indicating synergistic activity. Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo. Summary/Conclusion: In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL.

Activation of RAS/MAPK pathway confers MCL-1 mediated acquired resistance to BCL-2 inhibitor venetoclax in acute myeloid leukemia

Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2–selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

Chimeric antigen receptor (CAR) T-cell treatment for mantle cell lymphoma (MCL).

Mantle cell lymphoma (MCL) is a rare B-cell malignancy that remains challenging to treat with high rates of relapse. Frontline strategies range from intensive chemotherapy followed by consolidation with autologous stem cell transplant (ASCT), to less-intensive therapies including combination regimens. The treatment landscape for relapsed patients includes Bruton tyrosine kinase (BTK) inhibitors among other targeted treatments. Novel agents such as the selective BCL2 inhibitor venetoclax showed high response rates when used as monotherapy for refractory relapsed MCL. The rituximab, bendamustine, and cytarabine (R-BAC) regimen, while response rates were high, were not durable. Chimeric antigen receptor (CAR) T-cell products targeting CD19 have been efficacious in relapsed and refractory MCL patients. Brexucabtagene autoleucel (brexu-cel, formerly KTE-X19) was approved by US Food and Drug Administration (FDA) in July, 2020, for treatment of refractory and relapsed MCL. This article provides an overview for the available management strategies for relapsed MCL and examines the role of CAR T-cell in the current and future treatment of MCL.

AZD4573 Effectively Induces Apoptosis in r/r MCL As a Monotherapy or in Combination with Acalabrutinib

Mantle cell lymphoma (MCL) is an aggressive form of NHL where frequent relapse following standard therapies remains a serious concern, even for promising new treatments such as combinations of a BTK inhibitor with the selective Bcl2 inhibitor venetoclax. Previous studies have also shown that MCL cells develop resistance through tumor microenvironment interactions that increase levels of Mcl1, BclxL, and/or Bfl1. Given the ability of CDK9 inhibition to deplete Mcl1 and Bfl1 (Boiko et al 2021), we explored the potential of clinical-stage inhibitor AZD4573 to induce apoptosis in MCL cell lines and PDX models as a monotherapy or in combination with acalabrutinib. Currently, AZD4573 is being evaluated as a monotherapy in a first-in-human study for relapsed or refractory hematological malignancies (NCT03263637) as well as in combination with the BTK inhibitor, acalabrutinib, in patients with non-Hodgkin lymphoma (NHL) (NCT04630756).CDK9 is a serine/threonine kinase that mediates transcription elongation via phosphorylation of serine 2 of the RNA polymerase II carboxyl-terminal domain (pSer2-RNAP2). As previously shown, potent and selective inhibition of CDK9 by AZD4573 results in reduction of pSer2-RNAP2 levels leading to preferential depletion of labile proteins, including the Bcl2 family anti-apoptotic proteins Mcl1 and Bfl1 (as well as other known oncoproteins like Myc). This in turn drives rapid induction of apoptosis in a broad range of preclinical cancer models, particularly those derived from hematologic malignancies (Cidado et al 2020).Here, we used 7 MCL cell lines and 1 PDX organoid to assess the rapid apoptogenic potential of AZD4573 in vitro. Cleaved caspase-3 (CC3), a hallmark of apoptosis, was measured immediately following acute treatment (6h) using Caspase-Glo 3/7. Four models were sensitive to CDK9 inhibition (EC 50 < 100nM; max. CC3 > 50%) while 1 cell line exhibited intermediate sensitivity (EC 50 < 100nM; max. CC3 < 50%) and 3 others were resistant (EC 50 > 100nM; max. CC3 < 50%). Regardless of sensitivity, AZD4573 caused a dose- and time-dependent reduction of pSer2-RNAP2, Mcl1, and Myc, consistent with our prior reports.Most MCL cell lines are not responsive to BTK inhibition and, therefore, did not show combination benefit with AZD4573. We, therefore, chose to evaluate the in vivo activity of AZD4573 +/- acalabrutinib in 3 disseminated r/r MCL PDX models. In DFBL-44685, an acalabrutinib-unresponsive model harboring a CARD11 mutation, AZD4573 showed moderate activity, reducing MCL cells in all compartments analyzed (peripheral blood, bone marrow, and spleen) by >40% two weeks into treatment and increasing overall survival benefit (P<0.01). In an acalabrutinib-responsive MCL PDX, DFBL-96069, the combination of AZD4573 with acalabrutinib reduced tumor burden in all compartments analyzed by >80% and significantly increased survival over vehicle as well as monotherapy treatments (P<0.001). The third model, DFBL-98848, expresses high Bfl1. In this model, AZD4573 monotherapy reduced disease burden in all compartments analyzed by >50% and significantly increased survival over vehicle (P<0.001). Additionally, AZD4573 plus acalabrutinib further reduced disease burden and increased survival in this model compared to AZD4573 alone, although this did not reach statistical significance.Our findings show that targeting CDK9 with AZD4573 can effectively induce apoptosis in a range of MCL cell lines and PDX models, including acalabrutinib-sensitive and -insensitive models as well as those expressing high levels of Bfl1. In 3 r/r MCL PDX models, single agent AZD4573 significantly reduced the tumor burden in the peripheral blood, bone marrow, and spleen of the affected mice, resulting in increased survival. Combination of AZD4573 with acalabrutinib resulted in greater anti-tumor activity than either monotherapy. Altogether, these data suggest that AZD4573, alone or in combination with acalabrutinib, could be an effective therapy for patients with r/r MCL. [Display omitted] DisclosuresBoiko: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Andersen: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Liang: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Dowdell: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Reimer: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Drew: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Townsley: AstraZeneca: Current Employment, Current equity holder in publicly-traded company. Cidado: AstraZeneca: Current Employment, Current equity holder in publicly-traded company.

Diverse Mechanisms of Resistance to Decitabine and Venetoclax Therapy in Newly Diagnosed and Relapsed/Refractory AML Inferred By Transcriptome Analysis

Diverse Mechanisms of Resistance to Decitabine and Venetoclax Therapy in Newly Diagnosed and Relapsed/Refractory AML Inferred By Transcriptome Analysis

Adding venetoclax to fludarabine/busulfan RIC transplant for high-risk MDS and AML is feasible, safe, and active

AbstractAdding the selective BCL-2 inhibitor venetoclax to reduced-intensity conditioning chemotherapy (fludarabine and busulfan [FluBu2]) may enhance antileukemic cytotoxicity and thereby reduce the risk of posttransplant relapse. This phase 1 study investigated the recommended phase 2 dose (RP2D) of venetoclax, a BCL-2 selective inhibitor, when added to FluBu2 in adult patients with high-risk acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and MDS/myeloproliferative neoplasms (MPN) undergoing transplant. Patients received dose-escalated venetoclax (200-400 mg daily starting day −8 for 6-7 doses) in combination with fludarabine 30 mg/m2 per day for 4 doses and busulfan 0.8 mg/kg twice daily for 8 doses on day −5 to day −2 (FluBu2). Transplant related–toxicity was evaluated from the first venetoclax dose on day −8 to day 28. Twenty-two patients were treated. At study entry, 5 patients with MDS and MDS/MPN had 5% to 10% marrow blasts, and 18 (82%) of 22 had a persistent detectable mutation. Grade 3 adverse events included mucositis, diarrhea, and liver transaminitis (n = 3 each). Neutrophil/platelet recovery and acute/chronic graft-versus-host-disease rates were similar to those of standard FluBu2. No dose-limiting toxicities were observed. The RP2D of venetoclax was 400 mg daily for 7 doses. With a median follow-up of 14.7 months (range, 8.6-24.8 months), median overall survival was not reached, and progression-free survival was 12.2 months (95% confidence interval, 6.0-not estimable). In patients with high-risk AML, MDS, and MDS/MPN, adding venetoclax to FluBu2 was feasible and safe. To further address relapse risk, assessment of maintenance therapy after venetoclax plus FluBu2 transplant is ongoing. This study was registered at clinicaltrials.gov as #NCT03613532.